Project Summary Obesity increases the risk and adverse prognosis of postmenopausal estrogen receptor-positive (ER+) breast cancer. After menopause, estradiol falls and estrone is produced largely in fat by aromatase. Obesity women have high estrone, and both obesity and high estrone, but not estradiol, increase ER+ breast cancer risk after menopause. Obesity mediates chronic inflammation through NF-?B driven cytokine expression. We showed contact between invading breast cancer cells and obese adipose tissue induces pro-inflammatory cytokines in both cell types that stimulate cancer stem cells (CSC) and drive metastasis. Our data suggest that cytokine induction after breast fat:cancer cell contact is estrogen:ER dependent, since blocking estrogen synthesis with the aromatase inhibitor, letrozole, reduced cytokine induction upon co-culture. While estradiol is known to oppose NF-?B mediated inflammation, the role of estrone in inflammation is not known and may differ from that of estradiol. We will study how estrone and estradiol, and changes in the ratios thereof before and after- menopause, may influence NF-?B activity and the pro-inflammatory state in obese postmenopausal women. We hypothesize that increased estrone:estradiol ratio after menopause shifts ER from an NF-?B co- repressor to a co-activator to up-regulate cytokines in obese adipocytes and cancer cells that drive CSC expansion and metastasis. We also posit that enzymes that convert estradiol to estrone may contribute to the poor outcome of ER+ cancers in obesity. Aim 1 will test if estradiol:ER decreases, estrone:ER increases or different estradiol:estrone ratios alter ER effects on NF-?B mediated induction of pro-inflammatory cytokine drivers of CSCs in ER+ breast cancer cells. To test in Aim 2 if estrone cooperates with obesity to drive ER+ breast cancer growth, we will co-culture human ER+ breast cancer cells with mammary adipocytes from women with BMI +/- aromatase inhibition and test effects on cytokine levels and CSC. We will also implant syngeneic breast cancers into lean or obese wild-type or aromatase knock-out mice to elucidate if host estrone mediates tumor promoting effects of obesity. Aim 3 will test if overexpression of HSD17B14, that converts estradiol to estrone, increases ER+ breast cancer cytokine expression and CSC in vitro, and tumor initiation and metastasis in vivo. A better understanding of the roles of estradiol and estrone may lead to new strategies for breast cancer prevention and treatment, and to changes in hormone replacement therapies. Shifting toward a higher estradiol: estrone ratio in serum or in breast cancer cells may prove to have therapeutic potential. This SUPPLEMENT will test if estrone is more effective in stimulating ER+ breast cancer patient derived xenografts than estradiol. This will test our original hypothesis and help meet objectives of AIMS 1 & 2. It may also elucidate why ER+ breast cancer PDX have been so difficult to establish in the past. This work will also increase the number of ER+ PDX models for the research community to use for therapeutic research.